The thyroid peroxidase (TPO), the enzyme responsible for the iodination of thyroglobulin and biosynthesis of thyroid hormones, is an integral membrane heme-glycoprotein. TPO has been solubilized and partially purified using nonionic detergents. Preliminary studies suggested that TPO requires lipid for full enzymatic activity. Although much is known about the enzymatic activity of TPO, there is no information about the events in the biosynthesis of TPO, including post-translational modifications (proteolytic cleavage, disulfide linkage formation and glycosylation), assembly of putative subunits, and insertion of TPO into membranes. As an initial step in the elucidation of these mechanisms, we are preparing antibodies directed against the partially purified porcine TPO. Rabbits are being immunized with either detergent or trypsin solubilized porcine TPO. The anti-TPO antibody will be detected by specific inhibition of porcine TPO activity as determined by the guaiacol peroxidation assay and the iodination of thyroglobulin. The antisera will be used to purify further TPO, to detect the translation products containing the putative subunits of TPO directed by thyroid mRNA, and to detect endogenously (35S)-methionine labeled TPO. TPO, like many other thyroid proteins, is regulated by thyroid-stimulating hormone (TSH). These investigations will afford us an opportunity to study the effects of TSH on transcriptional, post-transcriptional, translational, and post-translational events in the biosynthesis of TPO that will be performed in conjunction with a newly developed thyroid cell culture system.